Horseradish peroxidase (HRP) was effectively entrapped in a novel composite membrane of poly(vinyl alcohol) and regenerated silk fibroin. IR spectroscopy was employed to characterize the structure of the composite membrane and scanning electron microscopy was used to visualize the morphology of the composite membrane. HRP was utilized to amplify the amperometric response of the sensor by catalyzing reduction of hydrogen peroxide via nickelocene, an electron transfer mediator between immobilized HRP and a glassy carbon electrode. The influence of various experimental parameters such as pH, temperature and applied potential were explored for optimum analytical performance. The sensor possessed a variety of characteristics including good sensitivity, rapid response time and a low detection of limit of 0.1 mu mol dm(-3), which arose from the efficiency of electron transfer between immobilized HRP and the electrode via nickelocene.